Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.     

Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.

We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases.     

Interferon alpha-induced apoptosis in tumor cells is mediated through the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway

Interferon (IFN) alpha induces a caspase-dependent apoptosis that is associated with activation of the proapoptotic Bak and Bax, loss of mitochondrial membrane potential, and release of cytochrome c. In addition to the onset of the classical Jak-STAT pathway, IFNalpha also induced phosphoinositide 3-kinase (PI3K) activity. Pharmacological inhibition of PI3K activity by Ly294002 disrupted IFN-induced apoptosis upstream of mitochondria. Inhibition of mTOR by rapamycin or by overexpression of a kinase dead mutant of mTOR, efficiently blocked IFNalpha-induced apoptosis. A PI3K and mTOR-dependent phosphorylation of p70S6 kinase and 4E-BP1 repressor was induced by IFNalpha treatment of cells and was strongly inhibited by Ly294002 or rapamycin. The activation of Jak-STAT signaling upon IFNalpha stimulation was not affected by abrogating PI3K/mTOR pathway. Neither was the expression of several IFNalpha target genes affected, nor the ability of IFNalpha to protect against virus-induced cell death affected by inhibition of the PI3K/mTOR pathway. These data demonstrate that an intact PI3K/mTOR pathway is necessary for the ability of IFNalpha to induce apoptosis, whereas activation of the Jak-STAT pathway alone appears to be insufficient for this specific IFNalpha-induced effect.     

The eukaryotic initiation factor 5A is involved in the regulation of proliferation and apoptosis induced by interferon-alpha and EGF in human cancer cells

Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.     

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-alpha effects in inducing apoptosis on human cancer cells through the generation of oxidative stress

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.     

Sphingosine 1-phosphate activates Erk-1/-2 by transactivating epidermal growth factor receptor in rat-2 cells

We investigated a signaling pathway leading to activation of extracellular signal-regulated protein kinase (Erk) 1 and 2 in Rat-2 cells stimulated with sphingosine 1-phosphate (S1P). S1P treatment transiently activated Erk-1/-2 in a dose-dependent manner, and its activation was blocked by pertussis toxin, expression of RasN17, or inhibition of Raf or MEK-1/-2. S1P-induced activation of Erk-1/-2 was also suppressed by the inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with the specific inhibitor AG1478, suggesting that activation of EGF receptor tyrosine kinase was involved in the signaling pathway. S1P-induced Erk-1/-2 activation was enhanced up to 2-fold by inhibiting protein kinase C (PKC) with GF109203X, and PKC inhibition in the absence of S1P treatment also activated Erk-1/-2. The stimulatory effects of Erk-1/-2 activation by PKC inhibition was blocked by treating cells with AG1478, suggesting the involvement of PKC in the regulation of EGF receptor tyrosine kinase activation that leads to Erk-1/-2 activation. Together, these results suggest that S1P activates the EGF receptor through a PKC-dependent pathway that links Ras signaling to the activation of Erk-1/-2 in Rat-2 cells.     

C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A

Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.     

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNalpha-induced apoptosis

Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2'-5' oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2'-5' oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNalpha-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNalpha-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNalpha and partially reverses IFNalpha-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt.     

Electromagnetic fields at mobile phone frequency induce apoptosis and inactivation of the multi-chaperone complex in human epidermoid cancer cells

The exposure to non-thermal microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, affects the refolding kinetics of eukaryotic proteins (Mancinelli et al., 2004). On these basis we have evaluated the in vivo effect of MW-EMF in human epidermoid cancer KB cells. We have found that MW-EMF induces time-dependent apoptosis (45% after 3 h) that is paralleled by an about 2.5-fold decrease of the expression of ras and Raf-1 and of the activity of ras and Erk-1/2. Although also the expression of Akt was reduced its activity was unchanged likely as a consequence of the increased expression of its upstream activator PI3K. In the same experimental conditions an about 2.5-fold increase of the ubiquitination of ras and Raf-1 was also found and the addition for 12 h of proteasome inhibitor lactacystin at 10 microM caused an accumulation of the ubiquitinated isoforms of ras and Raf-1 and counteracted the effects of MW-EMF on ras and Raf-1 expression suggesting an increased proteasome-dependent degradation induced by MW-EMF. The exposure of KB cells to MW-EMF induced a differential activation of stress-dependent pathway with an increase of JNK-1 activity and HSP70 and 27 expression and with a reduction of p38 kinase activity and HSP90 expression. The overexpression of HSP90 induced by transfection of KB cells with a plasmid encoding for the factor completely antagonized the apoptosis and the inactivation of the ras --> Erk-dependent survival signal induced by MW-EMF. Conversely, the inhibition of Erk activity induced by 12 h exposure to 10 mM Mek-1 inhibitor U0126 antagonized the effects induced by HSP90 transfection on apoptosis caused by MW-EMF. In conclusion, these results demonstrate for the first time that MW-EMF induces apoptosis through the inactivation of the ras --> Erk survival signaling due to enhanced degradation of ras and Raf-1 determined by decreased expression of HSP90 and the consequent increase of proteasome dependent degradation.     

Ras effector switching promotes divergent cell fates in C. elegans vulval patterning

The C. elegans vulva is patterned by epidermal growth factor (EGF) activation of Ras to control 1° fate, and 1° fate induces antagonistic Notch-dependent 2° fate. Furthermore, a spatial EGF gradient, in addition to inducing 1° fate, directly contributes to 2° fate via an unknown pathway. We find that in addition to its canonical effector, Raf, vulval Ras utilizes an exchange factor for the Ral small GTPase (RalGEF), such that Ras-RalGEF-Ral antagonizes Ras-Raf pro-1° fate activity. Consistent with its restricted expression pattern, Ral participates in EGF pro-2° activity. Thus, we have delineated a Ras effector-switching mechanism whereby position within the morphogen gradient dictates that Ras effector usage is switched to RalGEF from Raf to promote 2° instead of 1° fate. Our observations define the utility of Ras effector switching during normal development and may provide a possible mechanistic basis for cell and cancer-type differences in effector dependency and activation.     

An interferon-alpha-induced tethering mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu protein

Type 1 interferon (IFN) inhibits the release of HIV-1 virus particles via poorly defined mechanisms. Here, we show that IFNalpha induces retention of viral particles on the surface of fibroblasts, T cells, or primary lymphocytes infected with HIV-1 lacking the Vpu protein. Retained particles are tethered to cell surfaces, can be endocytosed, appear fully assembled, exhibit mature morphology, and can be detached by protease. Strikingly, expression of the HIV-1 Vpu protein attenuates the ability of human cells to adhere to, and thereby retain, nascent HIV-1 particles upon IFNalpha treatment. Vpu also counteracts the IFNalpha-induced retention of virus-like particles assembled from the Ebola virus matrix protein. Furthermore, levels of IFNalpha that suppress replication of Vpu-defective HIV-1 have little effect on wild-type HIV-1. Thus, we propose that HIV-1 expresses Vpu to counteract an IFNalpha-induced, general host defense that inhibits dissemination of enveloped virions from the surface of infected cells.     

Regulation of Akt by EGF-R inhibitors,a possible mechanism of EGF-R inhibitor-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine and a potent inducer of apoptosis. Epidermal growth factor (EGF) signaling is well known to involve in tumor survival and overexpression of EGF receptor (EGF-R) attributes to decreased responsiveness to many available therapies in cancer treatment. We investigated whether EGF-R inhibitors enhance TRAIL-induced apoptosis. We exposed A549 cells to Genistein, PD153035, and PD158780 for 12h and then treated with recombinant TRAIL protein. TRAIL alone induced 25% cell death after a 3-h treatment, but in cells pretreated with EGF-R inhibitors, TRAIL induced cell death to more than 70% after 3h treatment. Genistein enhanced TRAIL-induced apoptosis in a time- and dose-dependent manner. Western blot analyses showed that pretreatment with Genistein down-regulated the protein levels of total Akt and phosphorylated active Akt. Genistein also decreased the protein level of Bcl-XL that is regulated by Akt. These molecules are well characterized to act against induction of apoptotic cell death. Therefore, our data suggest that EGF-R inhibitor may sensitize A549 cells to TRAIL-induced apoptosis by regulating expression of these proteins. EGF-R inhibitors may play an important role in the anti-cancer activity of TRAIL protein, especially in TRAIL-resistant tumors that arise by expressing constitutively active Akt.     

Interferon alpha induces cell death through interference with interleukin 6 signaling and inhibition of STAT3 activity

In multiple myeloma, which commonly depends on interleukin 6, IL-6, survival signaling induced by this cytokine is largely mediated by activation of STAT3. Interferon alpha (IFNalpha) treatment of cell lines derived from multiple myeloma or of myeloma tumor cells ex vivo leads to apoptosis. In this study we demonstrate that IFNalpha treatment of the two myeloma cell lines, U266-1984 and U-1958, results in the decrease of STAT3 activity as demonstrated by a diminished STAT3/3 DNA-binding activity and the shift from STAT3/3 towards STAT1/1 and STAT3/1 complexes in EMSA, leading to the down-regulation of known STAT3 target genes such as Bcl-X(L), Mcl-1 and survivin. Ectopic expression of a form of STAT3, STAT3C, rescued U266-1984 cells from IFNalpha-induced apoptosis. IFNalpha promoted sustained accumulation of tyrosine phosphorylated STAT3C in the nucleus and a prolonged DNA binding of the STAT3/3 homodimers in EMSA. The shift towards a sustained STAT3 response in IFNalpha-treated STAT3C-transfected cells led to a hyper-induction of Bcl-2 and Mcl-1 proteins. Thus our data demonstrated that IFNalpha is able to interfere with IL-6 signaling by inhibiting STAT3 activity and that the abrogation of STAT3 activity accounts for the ability of IFNalpha to induce apoptosis in myeloma cells.     

Calcium/calmodulin-dependent protein kinase II binds to Raf-1 and modulates integrin-stimulated ERK activation

Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1.     

Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni. Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. (125)I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 x 10(-9) M) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.